Mutant proteins and methods for their production

ABSTRACT

The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallisation studies and also in screening to identify compounds for use in drug discovery and therapy.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/693,461, filed Nov. 25, 2019, which is a continuation of U.S. patent application Ser. No. 15/893,867, filed Feb. 12, 2018, which is a continuation of U.S. patent application Ser. No. 14/776908, filed Sep. 15, 2015, which is a national stage filing under 35 U.S.C. § 371 of international application PCT/GB2014/050757, filed Mar. 13, 2014, which was published under PCT Article 21(2) in English, and claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional patent application No. 61/790,592, filed Mar. 15, 2013, the disclosure of each of which is incorporated by reference herein in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing which has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created on May 27, 2022, is named S1492.70000US02-SEQ-JRV, and is 10,065 bytes in size.

FIELD

The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallisation studies and also in screening to identify compounds for use in drug discovery and therapy.

BACKGROUND TO THE INVENTION

Structure determination of eukaryotic integral membrane proteins is challenging and techniques and strategies developed recently has underpinned GPCR crystallization, including the development of receptor-T4 lysozyme (T4L) fusions (1,2), conformational thermostabilisation of GPCRs (3-7), and the use of antibody fragments (8-10).

However, the key component for successful crystallization is the stability of the GPCR during purification and crystallization (11). Techniques such as the addition of high affinity ligands to receptor-T4L fusions, and systemic mutagenesis coupled to a thermostability assay (3, 4, 6, 7) have improved the stability of GPCRs in detergent solution, detergent-stability being an essential prerequisite to purification and crystallisation. The latter approach locks the receptor in a particular conformation which allows successful crystallization as described in WO2008/114020 and WO2009/071914, and also has the advantage that the crystal structure of the GPCR bound to ligands that bind only very weakly can be determined (12,13). As described in WO2009/071914 it was found that stabilising mutations identified in one GPCR could be transferred to another GPCR by aligning the amino acid sequence thus generating a second GPCR with increased stability. However the positions of the stabilising mutations were not located in a common motif or region but instread scattered throughout the GPCR (the turkey β1-adrenergic receptor, human adenosine receptor, rat neurotensin receptor and human muscarinic receptor).

In recent years most success in obtaining crystal structures of membrane proteins has been for bacterial proteins (14) since these are easier to overexpress using known techniques in Escherichia coli than eukaryotic membrane proteins (15, 16) and are more likely to exhibit stability in detergent solution. In contrast eukaryotic membrane proteins often have poor stability in detergent solutions which severely restricts the range of crystallisation conditions that can be explored. Although the structures of over 300 unique polytopic integral membrane proteins have been determined (blanco.biomol.uci.edu/), less than 10% are eukaryotic and approximately half were purified from natural sources and are stable in detergent solutions.

Transmembrane transporters are similar to GPCRs because they exist in at least two distinct conformations, with the substrate binding site accessible to either the extracellular environment (outward-open) or to the cytoplasm (inward-open), with a number of potential intermediate occluded states where the substrate cannot dissociate to either side of the membrane (40). Indeed, the structures of many bacterial transporters have been determined that fit into the above scheme and, at least in the case of Mhp1 (41) and LeuT (26) different conformations of the same transporter have also been described. Transmembrane transporters have been less widely studied than GPCRs but nevertheless are highly relevant in human physiology and disease. They represent valuable targets in drug discovery and development of therapeutics, for example the monoamine transporters are key targets for therapeutic intervention in a wide range of CNS disorders and as primary targets for drugs of abuse such as cocaine and amphetamines (17, 18). Two of the most widely prescribed drugs fluoxetine (Prozac) and omeprazole (Prilosec) target membrane transporters, and there is a need in the art to understand further the structure and function of transmembrane transporters and their role in disease to meet the demand for new therapies targeting CNS disorders.

Current methodologies for the crystallisation of transporters have relied on the identification of those transporters that are sufficiently stable for purification and crystallisation (19) which has allowed the structure determination of many transporters from different families, but the majority of the structures are of bacterial proteins (20). To fully understand inhibitor binding and the mechanism of transport of the mammalian transporters it is essential to determine their structures. However the mammalian transporters are difficult targets for structural studies due to low levels of functional expression and only a proportion of the expressed protein is correctly folded (21, 22). Heterologous expression of the cocaine-sensitive rat serotonin transporter (SERT), GABA (GAT) and norepinephrine (NET) is possible in baculovirus systems however functional expression levels are low and only a proportion of the expressed protein is correctly folded according to the binding of radiolabelled inhibitors (23, 24).

The SLC6 transporter is a sub-class of the neurotransmitter sodium symporter family (NSS) (25) and plays an important role in regulating neurotransmitter concentrations in the peripheral and central nervous system by re-uptake into the presynaptic nerve termini. Mammalian SLC6 is characterised by 12 transmembrane helices with a large extracellular loop between transmembrane helices 3 and 4 (TM3 and TM4) that is invariably N-glycosylated. Structural studies on this family of transporters has focused on bacterial homologues that are extremely stable, such as LeuT (26).

Lactose permease of E. coli (LacY) has been the focus of a number of studies relating to structure and functionality and the crystal structure has been solved using a mutant (C154G) which renders the protein unable to undergo the structural changes required for transport of sugar across the membrane. The mutation (Cys154 to Gly) causes a more compact structure and decreased conformational flexibility with improved thermostability and little tendency to aggregate. It was also observed that the conformational change caused little or no effect on ligand binding (38). These studies demonstrate that it is possible to obtain a conformationally thermostabilised transporter membrane protein by way of mutation which is suitable for crystallisation. (39). Although these studies are of importance for Lactose Permease they do not comment on the wider problem of how to reliably and efficiently solve the crystal structures of other transporter proteins and GPCRs. Therefore there is a need for a common strategy to produce conformationally stable proteins that have use in crystallisation studies and structure determination. Since the provision of conformationally stable mutants of transporters and/or GPCRs and subsequent screening is time consuming, there is a need for methods that are more efficient and reduce the time taken to produce a conformationally stable mutant.

In view of the difficulty in obtaining high quality crystal structures of mammalian proteins due to poor stability and expression and the low numbers of solved crystal structures, there is a need to produce new methods and techniques that overcome the above-mentioned problems, particularly for membrane transporters which as yet have not benefited from the intensity of research seen for GPCRs.

The cocaine-sensitive rat serotonin transporter (SERT) is a member of the SLC6 sub-class of the neurotransmitter sodium symporter family (NSS) and transports the neurotransmitter serotonin from synaptic spaces into presynaptic neurons thereby terminating the action of serotonin. SERT has been well characterised in terms of physiological function and is the target of many antidepressant medications, however its crystal structure remains to be determined. SERT is known to be unstable in detergent solution (27) making it a challenging target for thermostabilisation studies, however the availability of a high affinity radiolabelled ligand [¹²⁵I] RTI55 (β-CIT) allows discrimination between functional and misfolded protein making the protein a suitable candidate for thermostabilisation studies.

The inventors have applied the conformational thermostabilisation approach recently used for GPCRs to the cocaine-sensitive rat serotonin transporter (SERT) to improve conformational stability and tolerance in detergent. They found that when particular regions in or nearby the alpha helices of the transmembrane regions of integral membrane proteins are mutated, this results in a higher proportion of conformationally stable mutants. This discovery has significance for structure determination of other related membrane proteins since it is possible that mutations in specific regions of a membrane protein can be applied across a range of membrane proteins having similar three-dimensional structures, thereby improving the probability of obtaining conformationally stable mutants for use in crystallisation.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a mutant transmembrane protein which has increased conformational stability compared to its parent transmembrane protein, wherein the one or more mutations are located at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink.

According to a further aspect of the invention there is provided a method of selecting a mutated transmembrane protein comprising the steps of;

-   -   a) Providing one or more mutants of a parent transmembrane         protein wherein the mutations are at the interfaces between         transmembrane alpha-helices, or in a kinked region or in an         alpha-helix adjacent to a kink.     -   b) Contacting the mutated transmembrane protein with a ligand     -   c) Determining the thermostability of the mutated transmembrane         protein     -   d) Identifying those mutants that exhibit increased         conformational thermostability compared to the parent         transmembrane protein

According to a further aspect of the invention there is provided a method of producing a mutated transmembrane protein comprising carrying out the steps a) to d) and,

-   -   e) Identifying the position of one or more of the mutated amino         acid residues in those mutants that exhibit increased         conformational stability, and     -   f) Synthesising a mutant transmembrane protein which comprises         the mutated residues identified in step e).

According to a further aspect of the invention there is provided a method of selecting a binding partner of a mutated transmembrane protein, the method comprising the steps of

-   -   a) providing a mutant transmembrane protein which has increased         conformational stability and/or is functionally inactive         compared to its parent transmembrane protein, wherein the one or         more mutations are located at the interfaces between         transmembrane alpha-helices, or in a kinked region or in an         alpha-helix adjacent to a kink.     -   b) contacting the mutant transmembrane protein with one or more         compounds     -   c) determining whether the one or more compounds bind to the         mutant transmembrane protein     -   d) isolating one or more compounds.

According to a further aspect of the invention there is provided a mutant transmembrane protein obtained by the methods of the previous aspects.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B Optimisation of SERT expression by transient transfection in T-Rex-HEK293 cells.

(FIG. 1A) The amount of tetracycline used to induce cells transiently transfected with SERT-mCherry was tested to give good cell surface expression with minimal intracellular expression, as defined by confocal microscopy. Examples of cells depicted were either induced with 1.2 μg/ml tetracycline (left hand panel) or 0.8 μg/ml tetracycline (right-hand panel). Cells were transfected with 0.1 mg of plasmid DNA per 50,000 cells and were induced for 48 hours. (FIG. 1B) The DNA:transfection reagent ratio for maximal correctly localised expression of SERT. 50,000 cells were transfected with a range of DNA concentrations. Cells were induced with 0.8 μg/ml tetracycline for 48 hours, examined using a confocal microscope and the percentage of plasma membrane expression of SERT was estimated by eye.

FIGS. 2A-2B Development of a thermostability assay for SERT.

(FIG. 2A) The thermostability of [¹²⁵I]-RTI55-bound SERT was determined after solubilisation in three different concentrations of DDM (final concentrations in %): squares, 0.01%; triangles, 0.1%; circles, 1%. The difference in thermostability is likely a consequence of the degree of delipidation of the transporter that increases as the amount of detergent increases. Each data point (±SEM) was obtained in duplicate from an equivalent of 50,000 cells from a tetracycline-induced stable cell line T-Rex-SERT. (FIG. 2B) Thermostability assays of SERT-His10 expressed in the stable cell line T-Rex-SERT (circles) and SERT-mCherry transiently transfected into T-Rex-HEK293 cells (squares). Cells were solubilised with 0.1% DDM after the addition of 1 nM [¹²⁵I]-RTI55; the apparent Tm of both samples of SERT was 28° C. All the reactions contained the equivalent of 50,000 cells per data point. The results are from a single experiment performed in duplicate (±SEM).

FIGS. 3A-3C Thermostabilisation of SERT

(FIG. 3A) Comparison of [¹²⁵I]-RTI55 bound to 554 detergent-solubilised Ala/Leu mutants at either 4° C. or after heating at 30° C. for 30 minutes. The data relating to thermostability (the heated samples) have been normalized to the amount of wild-type SERT remaining after heating (40%; horizontal dashed line). The expression level of wild-type SERT is indicated by the vertical dashed line and the grey column (0-200 dpm) represents non-specific binding of [¹²⁵I]-RTI55 to the parental T-REx-HEK293 cell line (no SERT). Each data point represents binding to the equivalent of 50,000 cells measured in duplicate (estimated error is ±20% in dpm). (FIG. 3B) The optimally thermostabilised SAH mutants were engineered by combining the best thermostabilising mutations (as indicated) which resulted in an increased apparent T_(m) after solubilisation in 0.1% DDM. (FIG. 3C) Amino acid sequence of SERT (SEQ ID NO: 1) showing the positions of the thermostabilising mutations (hatched) that consistently gave >1° C. increase in T_(m) compared to wild-type SERT; residues in bold and hatched were used to stabilize either SAH6 and/or SAH7. N-linked glycosylation sites are indicated by the hexagons, a putative disulphide bond is shown as a grey line and the N-terminal and C-terminal fusion partners are shown (c-Myc tag and the fluorescent reporter protein mCherry) are indicated.

FIG. 4 Alternative amino acid residues for SERT thermostabilisation.

Amino acid residues already identified as being thermostabilising in the preliminary Ala/Leu scan were changed either to Ala, Gly, Val Leu or Ile (hatched). Where no functional SERT was detected, the amino acid to which the residue was mutated is given in the single letter code. Bars marked with an asterisk showed improved thermostability over the original mutation, but expression levels were less than 10% of the wild type SERT and therefore these mutations were not used further.

FIGS. 5A-5B Detergent stability of the thermostabilised mutants SAH6 and SAH7.

(FIG. 5A) Thermostability curves for [¹²⁵I]-RTI55-bound DDM-solubilised SAH6 (circles) and SAH7 (squares) compared to wild-type SERT (triangles). The apparent T_(m)s determined from the curves are: SAH6, 46° C.; SAH7, 44° C.; wild-type SERT, 28° C. All the data were collected in a single experiment with measurements performed in duplicate. (FIG. 5B) The stability of [¹²⁵I]-RTI55-bound SAH6, SAH7 and wild-type SERT was compared in 8 different detergents [¹²⁵I]-RTI55 was added to membranes (final concentration 1 nM) which were then solubilised for 30 min on ice in the following detergents (aliphatic chain length in parentheses, final detergent concentration in %): 0.1% DDM (C12), 1% DDM (C12), 0.4% DM (C10), 0.35% FC12 (C12), 0.3% LDAO (C12), 0.6% Hega-10 (C10), 0.5% NG (C9), 0.6% NM (C9), 0.83% OG (C8). The detergent-solubilised samples where then heated at 30° C. for 30 min before determining the amount of SERT remaining in relation to control (incubated on ice). Results are from a single experiment performed in duplicate (±SEM) with the equivalent of 50,000 cells per data point.

FIG. 6 Inhibitor and substrate affinities for SAH6 and SAH7 mutants compared with wild-type SERT.

Apparent K_(i) values were determined from competition binding curves (FIGS. 7A-7C) and are plotted as the change in affinities with respect to wild-type SERT (ΔpK_(i)). Competition assays were performed on membranes using a final concentration of 0.2 nM [¹²⁵I]-RTI55 and apparent K_(i) values determined using the following apparent K_(D) values for [¹²⁵I]-RTI55 binding (FIGS. 7A-7C): SAH6, 3.7±0.7 nM; SAH7, 1.2±0.5 nM; wild-type SERT, 3.7±2.2 nM. Error bars are proportional to the SEM from the original measurements.

FIGS. 7A-7C Competition curves for SAH6 and SAH7.

(FIG. 7A) Competition assays were performed on membranes using a final concentration of 0.2 nM [¹²⁵I]-RTI55 and apparent K_(i) values (FIG. 7B) were determined using the following apparent K_(D) values for [¹²⁵I]-RTI55 binding (FIG. 7C): SAH6, 3.7±0.7 nM; SAH7, 1.5±0.6 nM; wild-type SERT, 3.7±2.2 nM. All results were obtained from two independent experiments performed in duplicate (±SEM) . For all graphs: wild type SERT, triangles; SAH6, circles; SAH7, squares.

FIGS. 8A-8B Thermostabilised mutants do not transport SHT.

(FIG. 8A) [³H]5HT uptake assays were performed on tetracycline-induced stable T-REx cell lines expressing SAH6-GFP, SAH7-GFP, and SERT-GFP, with non-specific uptake determined upon addition of 10 mM cocaine and the results baseline corrected. The results are from a single experiment performed in triplicate (±SEM) with approximately 100,000 cells per data point. (FIG. 8B) Confocal microscope images of cells expressing transporter-GFP fusions used in the uptake assays in (FIG. 8A). No fluorescence was detected in parental T-REx-HEK293 cells. SAH6-GFP, SAH7-GFP and SERT-GFP were all capable of binding [¹²⁵I]-RTI55-binding with high affinity (FIGS. 7A-7C).

FIG. 9 Amino acid sequence alignment between Rattus norveqicus SERT and Aquifex aeolicus LeuT (19% identity).

Identical residues (*) and similar residues (.) are indicated below the aligned amino acid sequences (SERT, SEQ ID NO: 1; LeuT, SEQ ID NO: 2). The transmembrane alpha-helices in LeuT (as defined by the structure) are shown as grey shading, with unwound regions as a grey line, and other alpha-helices are shown as hatched bars. The positions of the 11 thermostable mutations are shown either as hatched (present in SAH6 and/or SAH7) or hatched and shaded (other thermostabilising mutations).

FIGS. 10A-10C Equivalent positions in LeuT of the thermostabilising mutations in SAH6.

A cartoon of LeuT (PDB code 3GWU) is shown in black and white (N terminus light shade, C-terminus dark shade) with bound leucine (hatched spheres), Na+ ions (spheres) and sertraline (sticks). Shaded spheres indicate amino acid side chains in LeuT that are equivalent to the thermostabilising mutations found in SAH6 (in parentheses): Leu25 (L99A); Ala195 (G278A); Gly416 (A505L). LeuT is shown either viewed from the extracellular surface of the membrane (FIG. 10A) or parallel to the membrane plane (FIG. 10B). Portions of the structures have been highlighted (boxes, FIG. 10C) to depict the position of the residues in relation to helix-helix interfaces.

FIGS. 11A-11C Equivalent positions in LeuT of the thermostabilising mutations in SAH7.

A cartoon of LeuT (PDB code 3GWU) is shown in black and white (N terminus light shade, C-terminus dark shade) with bound leucine (hatched spheres), Na+ ions (spheres) and sertraline (sticks). Shaded spheres indicate amino acid side chains in LeuT that are equivalent to the thermostabilising mutations found in SAH7 (in parentheses): Leu322 (L405A); Ile410 (P499A); Gly416 (A505L). LeuT is shown either viewed from the extracellular surface of the membrane (FIG. 11A) or parallel to the membrane plane (FIG. 11B). Portions of the structures have been highlighted (boxes, FIG. 11C) to depict the position of the residues in relation to helix-helix interfaces.

FIGS. 12A-12D Amino acid residues in LeuT equivalent to the thermostabilising mutations in SAH6. (FIG. 12A) The structure of LeuT (PDB code 3GWU) is depicted in black and white (N terminus, light shade; C-terminus, dark shade) with the side chains in equivalent positions to the thermostabilisation mutations in SAH6 shown as shaded spheres. The view is from the extracellular surface perpendicular to the membrane plane. (FIGS. 12B-12D) The mutations are found at helix-helix interfaces and often at the sites of kinks or unwound regions; (FIG. 12B) Gly416^(LeuT) (A505L^(SAH6)); (FIG. 12C) Leu25^(LeuT) (L99A^(SAH6)); (FIG. 12D) Ala195^(LeuT) (G278A^(SAH6)). Additional views of the mutations are in FIGS. 10A-10C and for details of SAH7 mutations, see FIGS. 11A-11C.

FIGS. 13A-13D Crystallisation of thermostabilised SERT

(FIG. 13A) Crystallisation drop viewed under white light. (FIG. 13B) The same area of the crystallisation drop viewed under UV light where protein crystals appear white on a dark background. (FIG. 13C) One crystal mounted on a loop for collection of X-ray diffraction patterns. (FIG. 13D) One X-ray diffraction pattern showing diffraction spots to 8 Å resolution (the ring shows the 7.5 Å diffraction limit).

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a mutant membrane protein which has increased conformational stability compared to its parent membrane protein, wherein the one or more mutations are located at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink.

References herein to “membrane protein” refer to a protein that is attached to or associated with a membrane of a cell or organelle. Examples of membrane proteins include GPCRs, T-cell receptors, growth factor receptors, transmembrane ion channels, transmembrane transporters, a ligand-gated transmembrane ion channel, a voltage-gated transmembrane ion channel, an enzyme, a carrier protein or an ion pump. Membrane proteins may comprise more than one polypeptide chain.

For example the membrane protein may be an integral membrane protein that is permanently integrated into the membrane and can only be removed by using detergents, non-polar solvents or denaturing agents that physically disrupt the lipid bilayer.

References to “transmembrane protein” herein refer to an integral membrane protein which extends from one side of the membrane through to the other side of the membrane, thus spanning the entire membrane.

References to “transmembrane transporters” herein refer to transmembrane proteins which are responsible for the transport of ions, small molecules or macromolecules across a membrane. Suitable transmembrane transporters include monamine transporters, for example the cocaine-sensitive rat serotonin transporter (SERT), the dopamine transporter (DAT) and the norepinephrine transporter (NET).

In one embodiment the membrane protein is an integral membrane protein or a transmembrane protein.

In another embodiment the membrane protein is a transmembrane transporter or a GPCR.

In another embodiment the transmembrane transporter is a monamine transporter.

In a further embodiment the transmembrane transporter is a member of the SLC6 sub-class of the neurotransmitter sodium symporter family (NSS).

In a further embodiment the transmembrane transporter is the cocaine-sensitive rat serotonin transporter (SERT).

The membrane protein may be derived from any source, for example a eukaryotic source, a prokaryotic source, or from cell-free systems.

In one embodiment the membrane protein is mammalian and derived from rat, mouse, rabbit, dog, or non-human primate, man, chicken or turkey.

Suitable GPCRs are well known in the art and include those listed in Foord et al (2005) Pharmacol Rev. 57, 279-288, incorporated herein by reference, (which is periodically updated at iuphar-db.org/DATABASE/ReceptorFamiliesForward?type=GPCR).

Reference to “mutant membrane protein” herein refers to a membrane protein with a different genotype to the wild type (or parent) membrane protein. Such a mutant may also result in a different phenotypic difference.

Reference to “Parent membrane protein” herein refers to a protein which retains a functional activity of the naturally occurring protein. Functional activity may be for example, ligand binding and/or the transport of ions, small molecules or macromolecules across the membrane. The parent membrane protein may be more or less conformationally stable than the mutant membrane protein.

Mutants of the membrane protein may be produced by any suitable method where each amino acid of the parent membrane protein is independently changed to a different amino acid residue. Molecular biological techniques for cloning and engineering genes and cDNAs, for mutating DNA, and for expressing polypeptides from polynucleotides in host cells are well known in the art as exemplified “Molecular cloning, a laboratory manual”, third edition, Sambrook, J. & Russell, D. W. (eds), Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY, incorporated herein by reference.

Mutations may be made in any part of the membrane protein, for example in the part which spans the membrane.

In one embodiment the mutant membrane protein is produced by alanine scanning mutagenesis, a technique well known in the art. Here each selected amino acid is replaced in turn with alanine to produce a series of mutants suitable for screening. If the selected amino acid is Ala it is replaced with Leu or Gly. Selected regions of the membrane protein may be chosen for alanine mutagenesis, for example amino acid residues 49 to 603 inclusive for SERT.

In another embodiment the mutant membrane protein is produced by random mutagenesis, which may be in the whole of the protein or in a selection part. Such techniques are well known in the art (Asubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York 2000).

In one embodiment the mutant membrane protein comprises one or more mutations located at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink. The mutant membrane protein may also comprise one or mutations which are at the sites of unwound regions of the alpha-helix.

References to “transmembrane alpha-helices” herein refer to the helical three-dimensional amino acid structure found within the transmembrane spanning domains of a membrane protein. Transmembrane alpha-helices may be in the form of a single alpha-helix spanning the membrane, a pair of helices or any number of helices passing through the membrane typically ranging from one to twenty-four with common arrangements typified by seven helices passing through the membrane, as in GPCRs, or twelve helices passing through the membrane as in many transporters.

Reference to “interfaces” between transmembrane alpha-helices refers to the regions which are between the strands of the alpha-helix as arranged in the membrane, also known as helix-helix interfaces. Specifically interfaces are defined as regions were the atoms of amino acids residues in two or more separate helices are within van der Waals radii of each other.

Reference to “kinked region” herein refers to the regions which represent a turn of the alpha-helix as arranged in the membrane such that as a result of the kink the helix no longer has a linear conformation but instead comprises a bend or curve.

In one embodiment the mutant membrane protein comprises one or more replaced amino acids compared to the parent membrane protein. In another embodiment the mutant membrane protein comprises two, three, four, five, six or seven replaced amino acids compared to the parent membrane protein. In a further embodiment the mutant membrane protein comprises between two and four replaced amino acids compared to the parent membrane protein. In a further embodiment the mutant membrane protein comprises three or four replaced amino acids compared to the parent membrane protein.

In another embodiment the mutant membrane protein is the cocaine-sensitive rat serotonin transporter (SERT) and comprises at least one of the mutations selected from P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A.

In another embodiment the mutant cocaine-sensitive rat serotonin transporter (SERT) and comprises two, three or four of the mutations selected from P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A.

In another embodiment the mutant membrane protein is the cocaine-sensitive rat serotonin transporter (SERT) and comprises at least one of the mutations selected from G278A, A505L, L99A and P499A.

In a further embodiment the mutant membrane protein is the cocaine-sensitive rat serotonin transporter (SERT) and comprises the mutations L99A, G278A and A505L.

In a further embodiment the mutant membrane protein is the cocaine-sensitive rat serotonin transporter (SERT) and comprises the mutations L405A, P499A and A505L.

In another embodiment the mutant membrane protein comprises at least one or more mutations which are at the corresponding amino acid positions of P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A as defined in the amino acid sequence of SERT shown in FIG. 9 .

By “corresponding amino acid position or positions” we mean the position in the amino acid sequence of SERT which aligns to the position in the amino acid sequence of the membrane protein when the sequence of SERT and a membrane protein are compared by alignment using, for example, MacVector and the Clustal W program. Such alignment techniques are known in the art and appreciated by the skilled person.

In a further embodiment the mutant membrane protein comprises at least one or more mutations which are within a window of amino acids either side of the of the position which corresponds to the amino acid positions of one or more of P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A.

A “window of amino acids” refers to a window of i plus or minus four amino acid residues where i is defined as the stabilising mutation.

The stability of the mutant membrane protein is compared to the parent or wild type membrane protein to establish if the presence of the one or more mutations results in an increase in conformational stability.

Reference to “conformational stability” herein refers to the conformation adopted by a membrane protein that results in an improved stability with respect to any one of biological activity of the membrane protein such as binding activity, a signalling pathway modulation activity, a transmembrane transporting activity or an enzyme activity.

Increased conformational stability may be observed when the mutant membrane protein is bound to a ligand. A ligand may function as an inhibitor, an agonist or an antagonist. Ligand binding may cause the mutant membrane protein to reside in a particular conformation, for example with respect to GPCRs, an agonist or antagonist conformation depending on whether the ligand functions as an agonist or antagonist. Thus the presence of the ligand may be considered to encourage the GPCR to adopt a particular conformation. As a function of the ability of membrane transporters to facilitate the vectorial movement of substrates across biological membranes, they can exist in two different conformations known as the outward-open conformation and the inward-open conformation. Ligand binding may influence which conformation the membrane transporter resides in. In this respect, ligand binding that blocks or prevents transport of substances across the membrane functions as an inhibitor. Antibodies, including fragments and derivatives thereof, may function as ligands and induce conformational change in GPCRs and membrane transporters. Antibodies may be specific for a particular conformation of a GPCR or membrane transporter.

In one embodiment the mutant membrane protein is a membrane transporter and is conformationally stabilised in a single conformation such as the outward-open conformation or the inward-open conformation when bound to a ligand.

In another embodiment the mutant membrane transporter is SERT and is conformationally stabilised in the outward-open conformation when bound to a ligand, for example RTI55 (β-CIT).

The ligand may be detectably labelled in accordance with techniques known in the art. For example the ligand is radiolabelled or conjugated to a fluorescent label.

The mutant membrane protein may have an increased stability to any one or more of heat, a detergent, a chaotropic agent and extreme of pH.

Increased stability to heat (i.e. thermostability) can be readily determined by measuring ligand binding or by using spectroscopic techniques such as fluorescence, CD or light scattering at a particular temperature. In one embodiment the thermostability of a mutant membrane protein is determined by measuring the T_(m) (the temperature at which 50% of the mutant membrane protein is inactivated under certain conditions for a given period of time (e.g. 30 minutes). Mutant membrane proteins having a higher thermostability have higher T_(m) values when compared to the parent membrane protein.

To determine increased stability to a detergent or a chaotrope, the mutant membrane protein is incubated for a defined time in the presence of a test detergent or a test chaotropic agent and the stability is measured using for example ligand binding or a spectroscopic method as discussed above.

Suitable detergents for solubilisation of the membrane protein and/or mutant membrane protein and for measuring conformational stability are known to the skilled person in the art and include for example, dodecylmaltoside (DDM), CHAPS, octylglucoside (OG) and many others.

To determine increased stability to an extreme of pH, a typical pH test would be chosen, for example, in the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).

In one embodiment the mutant transmembrane protein has increased conformational thermostability when compared to the parent membrane protein.

In another embodiment the mutant transmembrane protein has increased conformational thermostability compared to the parent membrane protein by at least 1° C.

According to a further aspect of the invention, there is provided a method of selecting a mutated transmembrane protein comprising the steps of;

-   -   a) Providing one or more mutants of a parent transmembrane         protein wherein the mutations are at the interfaces between         transmembrane alpha-helices, or in a kinked region or in an         alpha-helix adjacent to a kink.     -   b) Contacting the mutated transmembrane protein with a ligand     -   c) Determining the stability of the mutated transmembrane         protein     -   d) Identifying those mutants that exhibit increased         conformational stability compared to the parent transmembrane         protein.

Methods according to steps b) to d) are known in the art and described in WO2009/071914 incorporated herein by reference. It is appreciated that such methods are equally applicable to transmembrane transporters.

In one embodiment increased conformational stability is increased conformational thermostability.

In another embodiment increased conformational stability is determined in the presence of detergent. Methods of providing one or more mutants of a parent transmembrane protein wherein the mutations are at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink is described above.

Methods of determining thermostability are as described in the methods section and in example 2.

In one embodiment the mutated parent transmembrane protein is contacted with a ligand prior to detergent-solubilisation and measurement of thermostability. Alternatively the mutated parent transmembrane protein is contacted with a ligand after detergent-solubilisation.

In one embodiment mutants of a parent transmembrane protein with increased conformational thermostability compared to the parent transmembrane protein were identified as having an increase in stability of at least 1° C.

According to a further aspect of the invention there is provided a method of producing a mutated transmembrane protein wherein the mutations are at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink, comprising carrying out the steps a) to d) and,

-   -   e) Identifying the position of one or more of the mutated amino         acid residues in those mutants that exhibit increased         conformational stability, and

Synthesising a mutant transmembrane protein which comprises the mutated residues identified in step e).

Methods for identifying the positions of the mutated amino acids according to step e) are carried out by sequencing techniques known in the art.

According to a further aspect of the invention there is provided a method of selecting a binding partner of a mutated transmembrane protein, the method comprising the steps of

-   -   a) providing a mutant transmembrane protein which has increased         conformational stability and/or is functionally inactive         compared to its parent transmembrane protein, wherein the one or         more mutations are located at the interfaces between         transmembrane alpha-helices, or in a kinked region or in an         alpha-helix adjacent to a kink.     -   b) contacting the mutant transmembrane protein with one or more         compounds     -   c) determining whether the one or more compounds bind to the         mutant transmembrane protein     -   d) isolating one or more compounds.

The mutated transmembrane proteins which have been identified as having conformational stability have use in the identification of ligands which bind to the transmembrane protein when it is in a particular conformation. The provision of ligands which bind the transmembrane protein in a particular conformation are valuable tools for the development of agents for therapeutic use.

Methods of screening are known in the art and described in WO2008/004223 herein incorporated by reference. It is appreciated that such methods of screening are equally applicable to transmembrane transporters.

In one embodiment the mutant membrane protein is immobilised onto a solid support.

According to a further aspect of the invention there is provided a mutated transmembrane protein obtainable by the methods described herein.

Methods

All radiolabelled ligands were purchased from Perkin Elmer and detergents were from Anatrace.

Thermostability Assay

The thermostability of detergent-solubilised [¹²⁵I]-RTI55-bound SERT was determined as previously described for GPCRs (3, 4, 6, 7). Briefly, cells containing unpurified SERT were incubated with 1 nM [¹²⁵I]-RTI55 for 30 min on ice, which were then solubilised with detergent (DDM) on ice for 30 min before incubation at varying temperatures for 30 min. The radioligand bound to the membrane protein was separated from free radioligand by centrifugal gel filtration and the radioligand bound to the eluted transporter measured by liquid scintillation counting.

Radiolabelled Inhibitor Binding Assay

Saturation binding curves for membrane-bound SERT were obtained using a range of [¹²⁵I]-RTI55 concentrations from 0.13 nM to 160 nM in a 96-well plate format with non-specific binding being accounted for by incubating identical samples with 1 μM cocaine. The samples were incubated for 2 hours at 30° C. and then filtered on 96-well glass-fibre plates (Millipore) pre-treated with 200 μl 0.1% polyethyleneimine. The filters were washed three times with 200 μl ice-cold SERT buffer (100 mM NaCl, 20 mM Tris pH 7.4), dried for one hour at 50° C. prior to liquid scintillation counting. Competition binding assays were performed as above but a range of concentrations of unlabelled ligand was included and a final concentration of 0.2 nM [¹²⁵I]-RTI55 was used.

[³H]-5HT Uptake Assays

The [³H]-5HT uptake assays were performed with slight modifications to the method previously described (28). In brief, T-Rex293 cells and T-REx-SERT cells were plated onto poly-L-lysine-coated (1 mg/ml) 24-well plates, grown to 80% confluency, induced by the addition of 0.8 μg/ml tetracycline and grown for 48 hours. The growth medium was aspirated and the cells washed once with TB buffer (10 mM Hepes pH 7.5, 150 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂). The assays were performed at 25° C. using 1 million cells in 400 μl TB buffer and 2 μM [³H]-5HT and terminated 3 minutes after addition of substrate by three washes of ice-cold TB buffer containing 1 μM paroxetine or 10 μM cocaine. [³H]-5HT was released by rupturing the cells with 2% SDS, which was quantified by liquid scintillation counting. Non-specific uptake was defined as [³H]-5HT transport in the presence of 10 μM paroxetine or 10 μM cocaine.

EXAMPLES Example 1: cDNA Expression of SERT in HEK293 Cells

The construct C-myc-SERT-mCherry-BioHis10 was developed from the SERT cDNA in plasmid pCGT137 (23) and inserted into the mammalian cell expression vector pcDNA5/FRT/TO (Invitrogen), which was used for site directed mutagenesis and expression. Cells were induced for 48 hours with 1.2 μg/ml or 0.8 μg/ml tetracycline. 0.1-0.2 μg of plasmid per 50,000 T-Rex-HEK293 cells was identified as the optimal amount to ensure the majority of SERT was expressed at the cell surface as observed by confocal microscopy (FIG. 1A).

Stable cell lines expressing SAH6-GFP and SAH7-GFP in T-REx-293 cells were generated by selection with media containing 200 μg/ml zeocin.

Example 2: Development of a Thermostability Assay for SERT

Binding assays using an excess of [¹²⁵I]-RTI55 at a concentration of 1 nM (5 times the K_(D)) (23) showed that there were on average approximately 100,000 copies per transfected cell and that there were sufficient molecules of SERT in 50,000 cells per well of a 96-well plate to perform a single-point thermostability assay in duplicate. Thermostability was determined after solubilisation in three different concentrations of DDM (0.01%, 0.1% and 1%) (FIG. 2A).

Thermostability assays usually involved adding the radioligand to detergent solubilised membrane proteins (7), but SERT is only stable in digitonin (29) therefore a different assay was developed where an inhibitor was used to stabilse the transporter. This entailed adding [¹²⁵I]-RTI55 to the T-Rex-HEK293 cells followed by detergent solubilisation and then the thermostability assay (heating samples at various temperatures for 30 minutes). The apparent T_(m) was defined as the temperature where 50% of the transporter still bound the radiolabelled inhibitor (FIG. 2B). For [¹²⁵I]-RTI55-bound SERT, the apparent T_(m) was 28° C., regardless of how it was expressed in HEK293 cells (FIG. 2B). Note that a considerable proportion of this thermostability is attributable to the bound inhibitor, because the apparent T_(m) of DDM-solubilised SERT without bound [¹²⁵I]-RTI55 could not be measured. [¹²⁵I]-RTI55-bound SERT was also sensitive to the concentration of DDM present in the assays (FIGS. 2A-2B), with the apparent T_(m) decreasing as the concentration of detergent increases. The most reproducible results with the steepest thermostability curve were obtained with a final concentration of 0.1% DDM, so this was used in subsequent assays to determine the thermostability of SERT mutants. The assays were repeated using either [³H]-imipramine or [³H]-paroxetine under identical conditions, but the binding characteristics of either ligand in the presence of DDM were unsatisfactory.

Example 3: Generation of SERT Mutants

Systematic alanine-scanning mutagenesis was performed throughout SERT between amino residues 49 and 603, with each residue changed to alanine or, if the residue was already alanine, then it was changed to leucine. Mutants were generated by PCR using the QuikChange II methodology (Stratagene) using KOD Hot Start polymerase (Novagen). PCR reactions were transformed into XL1-competent cells (Stratagene). Mutations were combined by PCR as above, but using multiple primers. Each SERT-mCherry mutant was sequenced to ensure that only the desired mutation was present. A total of 554 mutants were constructed throughout the transmembrane domains and all loop regions (FIGS. 3A-3C).

The N-terminus and C-terminus were not mutated because these regions were predicted to be disordered and they are therefore unlikely to contribute to the thermostability of SERT. Plasmid DNA for each SERT-mCherry mutant was amplified using a Maxi-prep kit (Qiagen) and transiently transfected (GeneJuice, Novagen) into adherent mammalian T-REx-293 cells (50% confluent) grown in DMEM media supplemented with 10% tetracycline-free FBS and 5 μg/L blasticidin. Expression of mutants was induced by addition of 0.8 μg tetracycline/ml and incubation at 37° C. for 24 hours. Expression was assessed by fluorescence microscopy to ascertain whether the mutant was predominantly either at the plasma membrane or intracellular. The thermostability of each mutant was then determined using a single-temperature thermostability assay and compared to the thermostability of wild-type SERT. The sample was heated at 28° C. for 30 minutes and approximately 40% of wild-type SERT remained functional. Each batch of mutants tested contained wild-type SERI as a control so that the data between different experiments could be normalized (wild-type=40%). Analysis of the results (FIGS. 3A-3C) identified 34 mutations that appeared to improve the thermostability of SERT, but which did not decrease the levels of expression by more than 70%. Interestingly, there was no correlation between the levels of expression and thermostability of the mutants, in contrast to in GPCRs where a weak correlation was sometimes observed (r²=0.2) (11). Of the 34 mutations identified, full thermostability curves showed that 10 mutations improved the thermostability of SERT by at least 1° C. as shown in Table 1 below.

TABLE 1 Thermostability and expression data for the most thermostable Ala/Leu mutants. Expression (%) Cell surface Apparent T_(m) ΔT_(m) Mutation SERT = 100% expression (° C.) (° C.) P499A 65 ** 35 7 A505L 175 *** 34 6 G113A 42 ** 33 5 L99A 131 ** 32 4 G278A 39 * 31 3 A169L 31 ** 30 2 F311A 492 ** 30 2 G115A 327 *** 29 1 L405A 543 *** 29 1 L406A 87 * 29 1 The top 34 SERT mutants as estimated from the single point thermostability assay were re-tested using a 6-point thermostability curve to determine an accurate apparent T_(m) and expressed as an improvement in T_(m) (ΔT_(m)) assuming wild-type SERT had an apparent T_(m) of 28° C. Each mutant was also assessed for cell surface expression as determined by estimation by eye of fluorescence throughout the cell upon confocal microscopy: * low expression; ** as wild-type SERT, *** higher expression than wild-type SERT.

Of these 10 mutations, 7 were in the transmembrane helices and 3 in the extracellular loops (FIGS. 3A-3C). Further mutation of these Ala/Leu mutants to other amino acid residues did not significantly improve the thermostability of SERT (FIG. 4 ).

Combining the thermostabilising mutations in SERT was performed by a rational process previously described for the thermostabilisation of agonist-bound neurotensin receptor and adenosine A_(2A) receptor (3). The best four thermostabilising mutations (P499A, A505L, L99A, G113A) were each combined with each other to make a series of double mutants (Table 2). Of these mutants, the most thermostable were A505L+L99A (SAH4) and A505L+P499A (SAH5). These double mutants were then combined with the remaining mutants to make triple mutants (Table 2), with the most thermostable being SAH6 (A505L+L99A+G278A) and SAH7 (A505L+P499A+L405A) with apparent T_(m)s 16° C. and 18° C. higher than wild-type SERT (FIGS. 5A-5B). Further combinations of mutations did not improve significantly the thermostability of these mutants (Table 2), so SAH6 and SAH7 were identified as the best candidates for structural studies and were therefore characterized further.

TABLE S2 Thermostability of double and triple mutants. Apparent T_(m) SERT mutation in 0.1% DDM Wild type SERT None 28° C. Double mutants A505L + P499A (SAH5) 39° C. A505L + L99A (SAH4) 43° C. P499A + L99A 35° C. P499A + F311A — A505L + F311A 32° C. L99A + F311A 27° C. Triple mutants A505L + P499A + L405A (SAH7) 44° C. A505L + P499A + A169L 33° C. A505L + P499A + F311A 44° C. A505L + P499A + L99A — A505L + L99A + G278A (SAH6) 46° C. A505L + L99A + F311A 43° C. A505L + L99A + L405A 43° C. A505L + L99A + L406A 44° C. A505L + L99A + N101A 40° C. A505L + L99A + G115A 44° C. A505L + F311A + L99A 40° C. Quadruple A505L + P499A + L405A + F311A 44° C. mutants A505L + P499A + L405A + G115A 45° C. A505L + L99A + G278A + F311A 44° C. A505L + L99A + G278A + G115A 44° C. A505L + L99A + G278A + L405A 44° C.

Combinations of SERT mutants tested for the thermostabilisation of ¹²⁵I-RTI55-bound detergent-solubilised SERT (apparent T_(m)±1° C.).

Surprisingly, when the mutations were mapped to the LeuT structure, all the mutations in SAH6 and SAH7 are found at the interfaces between transmembrane α-helices and, more specifically, in either a kinked region or in an α-helix adjacent to a kink. The conserved nature of amino acid residues that have been mutated to improve thermostability of SERT suggests that similar mutations in related transporters such as for norepinephrine (NET) and dopamine (DAT) would also improve their thermostability, as has been observed when thermostabilising mutations have been transferred between closely related GPCRs (37).

Example 4: Characterisation of Optimally Stabilised Mutants SAH6 and SAH7

Radioligand binding assays were performed on the thermostabilised SERT mutants as described above. The affinity of SAH6 and SAH7 for [¹²⁵I]-RTI55 in saturation ligand binding assays was found to be largely unchanged with apparent K_(D)s of 3.8±0.1 nM and 1.3±0.1 nM, respectively, compared to 1.6 nM±0.1 nM for wild-type SERT (FIGS. 7A-7C). Competition binding assays (FIGS. 7A-7C) showed that SAH6 had an apparent affinity for cocaine 17-fold higher than wild-type SERT (FIG. 6 ), whilst there was a small decrease in affinity for ibogaine, imipramine, paroxetine and serotonin (4.5-, 6.7-, 90-, 2.9-fold, respectively). SAH7 showed a similar profile of binding, although the absolute values differed slightly (FIGS. 7A-7C, FIG. 6 ).

Both SAH6 and SAH7 were found to be more stable in short chain detergents that are suitable for crystallography (11) and hence possess the most valuable and useful characteristic of thermostabilised GPCRs. SAH6 and SAH7 were also more tolerant to short chain detergents than wild type SERT (FIGS. 5A-5B).

The affinity of [¹²⁵I]-RTI55 for both SAH6 and SAH7 is virtually identical to the wild-type transporter (23), which strongly supports the contention that the mutants are folded in a biologically relevant conformation. This is further supported by the cell surface expression of both mutants in stable cell lines expressing either SAH6 or SAH7, as misfolded SERT is retained in the endoplasmic reticulum (ER). Competition assays using both inhibitors and the substrate 5-HT provide further evidence on the likely conformation that has been stabilised. Both SAH6 and SAH7 bind cocaine with higher affinity than wild-type SERT. None of the mutations are in the region proposed to be the inhibitor binding site (30, 31) so in analogy to what has been previously observed in GPCRs, these data suggest that SAH6 and SAH7 have been stabilised in a ‘cocaine-bound’ conformation. As cocaine has been proposed to bind preferentially to the outward-open conformation of SERT (32), it is likely that both SAH6 and SAH7 are thermostabilised in an outward-open state. The decrease in binding affinity of both imipramine and paroxetine for SAH6 is consistent with this interpretation, as there are likely to be subtle differences between the binding of these inhibitors compared to RTI55, even though they are all proposed to bind to the outward-open state (33, 34).

Example 5: Functional Activity of SAH6 and SAH7 Mutants

A characteristic of transporters is obviously their ability to facilitate the vectorial movement of substrates across biological membranes. 5HT transport catalysed by SAH6 and SAH7 was therefore compared with the wild-type transporter in stable cell lines that robustly express the transporters on the cell surface of a tetracycline-inducible HEK293 cell line (35, 36).

Although both SAH6 and SAH7 were capable of binding inhibitors and the substrate serotonin, no significant transport of [³H]-5HT into the cell was observed in cell lines expressing cell surface-expressed SAH6 or SAH7, despite the presence of the mutants in the plasma membrane as defined by confocal microscopy (FIGS. 8A-8B) suggesting that the receptors are preferentially in one particular conformation. This is not due to alterations in the binding site for 5HT, because 5HT prevented [¹²⁵I]-RTI55 binding in competition assays although the affinity for 5HT was decreased by 1.5-2.9 fold. These data are consistent with the theory that both SAH6 and SAH7 are thermostabilised in a specific outward-open conformation.

Example 6: Mapping of Thermostabilising Mutations to LeuT

There is a growing body of data which suggests that the structure of SERT is very similar to that of the bacterial transporter LeuT. Structures of LeuT bound to antidepressant drugs have been determined, where the antidepressant drugs are also known to bind SERT (30, 31) and despite the large difference in binding affinities (nM compared to nM), has led to plausible models for how antidepressant drugs inhibit SERT. We have therefore mapped the thermostabilising mutations identified herein to the structure of LeuT bound to sertraline (FIGS. 9-11C, FIG. 12A-12D). Surprisingly it was found that all the mutations in SAH6 and SAH7 are found at the interfaces between transmembrane α-helices and. More specifically, in either a kinked region or in an α-helix adjacent to a kink, This suggests that mutations in specific regions of a protein can be applied across a range of membrane proteins having similar three-dimensional structures, thereby improving the probability of obtaining conformationally stable mutants for use in crystallisation.

Example 7: Expression and Crystallisation of Thermostabilised SERT

A stable HEK293-GnTI⁻ cell line was constructed that expressed the thermostabilised serotonin transporter (SERT) to high levels. On induction with tetracycline, thermostabilised SERT was expressed sufficiently to allow the purification of 2 mg of transporter from 10 L of cells using the detergent dodecylmaltoside. Crystals were produced by vapour diffusion methodology in 23% PEG400, 100 mM MES pH 5.9 and they diffracted isotropically to about 8 Å resolution.

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1.-24. (canceled)
 25. A mutant membrane protein which has increased conformational stability compared to its parent membrane protein, wherein the one or more mutations are located at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink.
 26. A mutant membrane protein according to claim 25 which is a T-cell receptor complex, a growth factor receptor, a ligand-gated transmembrane ion channel, a voltage-gated transmembrane ion channel, a transmembrane transporter, an enzyme, a carrier protein, or an ion pump.
 27. A mutant membrane protein according to claim 26 which is a transmembrane transporter; optionally wherein the transmembrane transporter is a member of the solute carrier proteins (SLC) family.
 28. A mutant membrane protein according to claim 27 which is a transmembrane transporter and a member of the neurotransmitter sodium symporter family (NSS).
 29. A mutant membrane protein according to claim 28 which is a member of the SLC6 subclass of the NSS family; optionally a mammalian SLC6 protein.
 30. A mutant membrane protein according to claim 29 wherein the mutant membrane protein is a norepinephrine transporter (NET) protein, a dopamine transporter (DAT) protein, or the a glycine transporter (GlyT) protein, or a serotonin transporter (SERT) protein.
 31. A mutant membrane protein according to claim 29 which is a monoamine transporter protein.
 32. A mutant membrane protein according to claim 31 which is a norepinephrine transporter (NET) protein, a dopamine transporter (DAT) protein, or a serotonin transporter (SERT) protein.
 33. A mutant membrane protein according to claim 25, wherein the mutant membrane protein is a serotonin transporter (SERT) protein; optionally a mammalian SERT protein .
 34. A mutant membrane protein according to claim 25, wherein the one or more mutations comprises three or more mutations.
 35. A mutant membrane protein according to claim 25, wherein each of the one or more mutations is i) an amino acid residue replaced with alanine or ii) if the amino acid residue was already alanine, then it is replaced with leucine.
 36. A mutant membrane protein according to claim 25, wherein the mutant membrane protein comprises at least one or more mutations which are within a window of amino acids either side of the position which corresponds to the amino acid positions of one or more of P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A as defined in the amino acid sequence of the serotonin transporter (SERT) shown in FIG. 9 ; wherein the window of amino acids refers to a window of i plus or minus four amino acid residues where i is defined as the stabilising mutation.
 37. A mutant membrane protein according to claim 25 which has increased conformational stability when compared to its parent to one or more of heat, a detergent, a chaotrope or an extreme of pH.
 38. A mutant membrane protein according to claim 25 which is bound to a ligand. 